Chromatin immunoprecipitation on native chromatin from cells and tissues

Introduction

In cells and tissues, the histone proteins that constitute the nucleosomes can present multiple post-translational modifications (Luger & Richmond, 1998), such as lysine acetylation, lysine and arginine methylation, serine phosphorylation, and lysine ubiquitination. On their own, or in combination, these covalent modifications on the core histones are thought to play essential roles in chromatin organisation and gene expression in eukaryotes (Hebbes et al ., 1994; O'Neill & Turner, 1995; Grunstein, 1998; Turner, 2000; Jenuwein & Allis, 2001). Importantly, patterns of histone modifications may be somatically conserved and can, thereby, maintain locus-specific repression/activity in defined lineages, or throughout development. Indirect immuno-fluorescence studies on cultured cells have been pivotal in unravelling the roles of histone modifications. These studies have been highly informative on the functions of specific histone modifications in, for instance, pericentric chromatin condensation (Peters, et al ., 2001; Maison, et al ., 2002) and X-chromosome inactivation (Heard et al ., 2001, Boggs et al ., 2002, Peters et al ., 2002) in mammals (H3 and H4 deacetylation, and H3-K9 methylation). However, particularly in mammalian model systems, it remains poorly understood how histone modifications are organised at specific chromosomal regions and genes. To address in detail what happens at specific sites in vivo, chromatin immuno-precipitation (ChIP) is the method of choice. Here, we describe how ChIP can be performed on native chromatin extracted from cells, or tissues, to analyse histone methylation and acetylation at specific chromosomal sites. In addition, we present different PCR-based methods that allow the analysis of a locus of interest in chromatin precipitated with antibodies to specific histone marks (see figure 1 for an overview of the described procedures). Should you require a literature reference, please, quote an earlier paper by our group, where these methodologies were originally described (Umlauf et al. , 2003).

Background information

Chromatin immuno-precipitation

Chromatin immuno-precipitation (ChIP) is performed by incubation of fractionated chromatin (input chromatin) with an antiserum directed against a chosen histone modification. As a general rule, there are two ways to obtain 'input' chromatin. Several groups in the field prepare 'cross-linked chromatin', for example, by photochemical cross-linking, or by chemically cross-linking proteins and DNA with specific substances, such as formaldehyde (Orlando, 2000). Formaldehyde cross-linking is particularly suitable for ChIP studies on histone modifications. However, usually only a small fraction of the chromatin is precipitated, and it relies on random shearing, which does not always produce small-enough chromatin fragments at the regions of interest. For this reason and to conduct experiments on fresh and frozen tissues, we and others have preferred to make use of 'native chromatin' (O'Neill & Turner, 1995; Gregory et al ., 2001). In these protocols (Figure 1), the chromatin is fractionated by incubation of purified nuclei with micrococcal nuclease (MNase), an enzyme that cleaves preferentially the linker DNA between the nucleosomes (Drew, 1984). Specifically, by performing partial digestions with MNase, it is possible to obtain native chromatin fragments of on average one to five nucleosomes in length (Figure 2). These oligo-nucleosome fragments are purified from the nuclei and are then used to perform ChIP. The choice of native, MNase-fractionated, chromatin as the input material for ChIP is advantageous, because the epitopes, recognised by the antibody, remain intact during the chromatin preparation. As a consequence, native chromatin tends to give higher levels of precipitation for a specific histone modification than formaldehyde cross-linked chromatin (Goto et al ., 2003). The ChIP protocol presented below describes in detail how to prepare and immuno-precipitate native chromatin. This protocol was adapted from methodology originally described by O'Neill and Turner (1995), and allows ChIP to be performed not only on chromatin from cultured cells, but also on freshly dissected and frozen tissues. It is adapted to the analysis of histone methylation and acetylation.

Although ChIP is presently the best-available methodology to analyse histone modification at specific chromosomal loci, it has several limitations. Firstly, unlike DNA methylation studies, ChIP does not allow analysis of histone modifications in individual cells, or on individual chromosomes. ChIP studies are always performed on populations of (cultured) cells, or on tissue samples comprising many cells. Although sequential precipitations with different antisera could be attempted, this method seems also not suitable to determine whether there are specific combinations of covalent modifications on individual histones at a given locus. Again, this is because many cells are used for chromatin purification and ChIP, and chromatin is usually fractionated into fragments that comprise multiple nucleosomes. Lastly, it should be noted that quantification of the levels of histone modifications at specific chromosomal loci is difficult to obtain by ChIP, because levels of precipitation do not depend simply on the local abundance of the modification studied. They also depend, to a great extent, on the quality of the prepared chromatin, and on how the chromatin was prepared (eg, native versus cross-linked), on the quality, specificity and concentration of the antiserum used for ChIP, and on the global abundance of the histone modification that is being studied. The later limitation of the technology is discussed in more detail in the footnotes below.

PCR-based analysis of precipitated chromatin

After ChIP, precipitated chromatin fractions are analysed by optical density (OD) reading and agarose gel electrophoresis to assess the quantity and quality of the precipitated chromatin. DNA is then extracted to allow analysis of the chromosomal site(s) of interest. In several earlier studies on locus-specific histone modifications, regions of interest were analysed by Southern hybridisation of slot blots (O'Neill & Turner, 1995; Hebbes et al ., 1988). More recently, however, quantitative amplification by polymerase chain reaction (PCR) has become the method of choice. Different PCR-based approaches can be used to determine how much DNA is precipitated at a site of interest. Although 'real-time' PCR amplification is often the preferred technique to quantify amounts of chromatin precipitated at specific loci, 'Duplex PCR amplification', which is the co-amplification of a fragment from the region of interest and a control fragment (e.g. the actin gene, or the tubulin gene), can also be used. Duplex PCR amplification has been successful in studies on the S. pombe mating-type loci and for analysis of imprinted mammalian genes (Figure 3), as it allows to detect relative levels of specific histone modifications along chromosomal domains (Gregory et al ., 2001; Norma et al ., 2001). Alternatively, in particular for allelic studies on dosage-compensation mechanisms, or on genomic imprinting in mammals, Single-Strand Conformation Polymorphisms (SSCP, Figure 4) (Orita et al ., 1989; Gregory & Feil, 1999), or similar strategies, such as 'Hot Stop PCR' (Uejima et al ., 2000), can be put to use to differentiate PCR products that represent the silent allele from those amplified from the active allele (Goto et al ., 2003; Fournier et al ., 2002). These different PCR-based approaches will be described in detail in the section on quantitative PCR analysis of precipitation chromatin.

Acknowledgements

We thank Richard I. Gregory for his help with the design of methodologies and Bryan M. Turner and Laura P. O'Neill (Birmingham, UK) for introducing us to immuno-precipitation on native chromatin. The HFSP, the CNRS, the Fondation pour la Recherche Médicale (FRM), and the Association pour la Recherche contre le Cancer (ARC) are acknowledged for grant support.

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Procedure

Nuclei Preparation from Tissues and Cells

To prevent chromatin degradation by endogenous nucleases and to keep the chromatin intact, all steps of the nuclei purification procedure should be performed on ice, or at 4°C (e.g. pre-cool the centrifuge rotors). In addition, dedicate one set of pipettes only for the preparation of nuclei, chromatin and ChIP analysis, to avoid contamination with non-genomic DNA (plasmids, PCR products, etc.).

Purification of Nuclei from Tissues

1. Dissect tissue (do not use more than 0.2g in total) and rinse it in cold PBS (see note 1 and note 2);
2. Homogenise tissue in a pre-chilled glass homogenizer with 5-10mL of ice-cold Buffer I, until no clumps of cells persist (about 10-20 strokes). Filter the cell suspension through 4 layers of muslin cheese cloth that have been moistened beforehand with 2mL of Buffer I;
3. Transfer the cell suspension to a 14mL polypropylene tube and spin cells down in a swing-out rotor (at 6000 g for 10 minutes, at 4°C);
4. Pour off the supernatant and re-suspend the cells in 2mL of ice-cold Buffer I. Then add 2 mL of ice-cold Buffer II (see note 3 and note 4), mix gently, and place on ice for 10 minutes.
5. Prepare two new 14mL polypropylene tubes containing each 8mL of ice-cold Buffer III. Carefully layer 2mL of each cell suspension (from step 4) on each 8mL sucrose cushion. Cover the tubes with a piece of Parafilm®;
6. Centrifuge in a pre-chilled swing-out rotor, at 10,000 g, for 20 minutes at 4°C. During this centrifugation step, the nuclei will form a pellet on the bottom of the tube, whereas the cytoplasmic components will remain in the top layer (see note 5);
7. Carefully take off the supernatant with a Pasteur pipette. This is a critical step, as the top solution (which contains the detergent IGEPAL CA-630®) should not come into contact with the nuclear pellet at the bottom of the tube. One way to achieve this, is to remove the supernatant in about three times, each time changing the Pasteur pipette (see note 5);
8. Re-suspend the nuclei pellet into 1mL of MNase digestion buffer and put on ice. Nuclei can, at this point, be counted by using a microscope slide for counting cells. The number of nuclei obtained per gram of tissue varies between different tissue types. For liver, for example, the above protocol yields ~2x10⁹ nuclei/gram of tissue. (Frozen tissues can be used for nuclei preparation as well (see note 6, note 7 and comment 1).

Nuclei Preparation from Cultured Cells

Culture 5x10⁷ to 5x10⁸ cells in the appropriate culture medium. Ensure that cells are not grown beyond semi-confluency.

1. Rinse cells in PBS, add 2mL of trypsin solution (for adhering cells only) and incubate at 37°C;
2. When trypsination is complete, stop the reaction by adding 5mL of culture medium to the cells;
3. Divide the cell suspension amongst two polypropylene 14mL tubes and spin cells down in a swing-out rotor (4000 g, 5 minutes at 4°C);
4. Pour off the supernatant and re-suspend the cells in 2mL of ice-cold Buffer I. Then add 2mL of ice-cold Buffer II (see note 3 and note 4), mix gently, and place on ice for 10 minutes;
5. Prepare two new 14mL polypropylene tubes containing each 8 mL of ice-cold Buffer III. Carefully layer 2mL of each cell suspension (from step 4) on each 8mL sucrose cushion. Cover the tubes with a piece of Parafilm®;
6. Centrifuge in a pre-chilled swing-out rotor, at 10,000 g, for 20 minutes at 4°C. During this centrifugation step, the nuclei will form a pellet on the bottom of the tube, whereas the cytoplasmic components will remain in the top layer (see note 5);
7. Carefully take off the supernatant with a Pasteur pipette. This is a critical step, as the top solution (which contains the detergent IGEPAL CA-630®) should not come into contact with the nuclear pellet at the bottom of the tube. One way to achieve this, is to remove the supernatant in about three times, each time changing the Pasteur pipette (see note 5);
8. Re-suspend the nuclei pellet into 1mL of MNase digestion buffer and put on ice. Nuclei can, at this point, be counted by using a microscope slide for counting cells. The number of nuclei obtained per gram of tissue varies between different tissue types. For liver, for example, the above protocol yields ~2x10⁹ nuclei/gram of tissue. (Frozen tissues can be used for nuclei preparation as well (see note 6).

MNase Fractionation and Purification of Chromatin

Dialysis tubing needs to be prepared before starting the purification of chromatin. Several batches can be prepared and stored at 4°C for several weeks.

Preparation of Dialysis Tubing

1. Cut the tubing into pieces of convenient length (10-20cm);
2. Boil the tubes for 10 minutes in 0.5L of tubing preparation solution I;
3. Rinse the tubes twice in distilled water;
4. Boil the tubes for 10 minutes in 0.5L of tubing preparation solution II;
5. Allow the tubes to cool down and store them in tubing preparation solution II at 4°C. Ensure that the tubes are entirely submerged;
6. Before use, wash the tubing twice inside and out with distilled water.

MNase Fractionation

1. Purified nuclei (as described the section titled Nuclei Preparation from Tissues and Cells) are re-suspended in 1mL of ice-cold MNase digestion buffer and placed on ice;
2. Aliquot the suspension in two 1.5mL Eppendorf tubes (500µl of re-suspended nuclei in each tube);
3. Add 1µl of MNase enzyme to each tube and mix gently;
4. Incubate the two tubes in a 37° water-bath for 6 and 9 minutes, respectively;
5. Add 20µl of stop solution;
6. Chill on ice.

Recovery of Soluble Chromatin Fractions

1. Centrifuge the 1.5mL tubes with the MNase-digested nuclei at 10,000 rpm (4°C) for 10 minutes to pellet the nuclei;
2. Transfer the supernatant into another 1.5mL tube and store at 4°C. This supernatant contains the first soluble fraction of chromatin, S1, which comprises small fragments only. Do not discard the pellet;
3. Carefully re-suspend the pellet in 500µl of dialysis buffer;
4. Close one side of the washed dialysis tubing (see section titled Purification of Nuclei from Tissues, step 6) with a universal closure clamp. Transfer the 500µL of re-suspended nuclei into the dialysis tube and close the other side with a second clamp;
5. Submerge the tube for 12-16 hours in 1-2L of dialysis buffer. Dialysis is performed at 4°C, in a beaker with constant mild stirring using a magnetic stirrer;
6. Transfer the dialysed nuclei into a 1.5mL Eppendorf tube;
7. Centrifuge for 10 minutes at 10,000 rpm, at 4°C, in a micro-centrifuge;
8. Transfer the supernatant in a new 1.5mL Eppendorf tube and store at 4°C. This is the second soluble chromatin fraction, S2, comprising larger fragments of chromatin, that were removed from the nuclei during the dialysis;
9. Re-suspend the pellet, which is the chromatin fraction P, in 50µl of dialysis buffer and store at 4°C.

Quality Control of Chromatin

1. Take the Optical Density (OD) of each fraction at 260nm;
2. In separate 1.5mL Eppendorf tubes, put 0.5µg of each fraction (S1, S2, and P);
3. Add 2µL of loading buffer, and 1µL of 10% SDS. Adjust the volume to 10µL and mix gently;
4. Load the samples onto a standard 1% (w/v) agarose gel (of about 10-15cm in length) in 1x TBE electrophoresis buffer, with the 100bp DNA ladder as a size control. Let the samples migrate at 2-3V/cm until the fastest blue marker in the loading buffer has migrated till about half way the gel;
5. Stain the gel for 30 minutes in a tray with 500mL of distilled H₂ O to which 10µg of Ethidium Bromide have been added;
6. Remove background staining from the gel, by rinsing for 15 minutes in distilled H₂ O;
7. Control the size of the chromatin fragments under an UV lamp, and take a photograph (see figure 2 for an example of typical S1 and S2 fractions). The pellet fraction, P, consists of chromatin fragments that are longer than 5 nucleosomes in length.

Chromatin Immuno-precipitation

When an antiserum is used for the first time, it should be important to verify that the histone modification, it is directed against, has become enriched in the antibody-bound fraction. This can be done by purifying the histone proteins from this fraction followed by electrophoresis through acid-urea-triton gels (O'Neill & Turner, 1995; Bonner et al ., 1980). After electrophoresis, proteins are Western blotted to nylon filters, which are immuno-stained with the antiserum, following standard procedures (Sambrook & Russell, 2001). An example of this procedure (Bonner et al ., 1980) is presented by Gregory et al . (2002), relative to a study on histone acetylation in ES cells and fibroblasts.

Incubation of Chromatin with Antiserum

1. Mix 10-20µg of the first (S1) and 10-20µg of the second (S2) chromatin fractions in a 1.5mL Eppendorf tube (see note 8);
2. Complete the volume to 1mL with ChIP incubation buffer;
3. Add 5-10µg of the antibody of choice;
4. Close the tubes and seal the lids with some Parafilm®. Rotate the tubes at 20-30 rpm for 12-16 hours at 4°C. During this incubation time the antibodies will bind to their specific epitopes;
5. Meanwhile, aliquots of Protein-A (G) Sepharose (see following section) can be prepared for the extraction of the immuno-precipitated chromatin (see section titled Extration of Immuno-precipitated Chromatin with Protein-a (G) Sepharose).

Preparation of Protein-A (G) Sepharose (see note 8 and note 9)

1. Weigh 0.25g of Protein-A (G) Sepharose beads into a 14mL polypropylene tube;
2. Add 1mL of sterile water to moisten the beads;
3. Wash with 10mL of sterile water and mix;
4. Centrifuge for 3 minutes at 1500 g in a swing-out rotor and discard the supernatant;
5. Repeat steps 3 and 4 four times;
6. Add 1mL of sterile water and re-suspend the beads;
7. Distribute 100µL aliquots in ten 1.5mL Eppendorf tubes. Store these aliquots at 4°C. They can be used for the extraction of antibody-bound chromatin from ChIP experiments.

Extraction of Immuno-precipitated Chromatin with Protein-A (G) Sepharose

1. Add 50µl of protein-A Sepharose to each tube after the immuno-precipitation (step 4 of incubation of chromatin with antiserum) (see note 9);
2. Let the tubes rotate at 20-30 rpm, for 4 hours, at 4°C;
3. Centrifuge at 1500 g in a swing-out rotor for 3 minutes;
4. Transfer the supernatant to a 2mL Eppendorf tube. This fraction contains the chromatin which did not link the antibody: the unbound fraction. To be stored on ice;
5. Re-suspend the Sepharose beads in 1mL of Washing buffer A;
6. Transfer the re-suspended beads to a 15mL Falcon tube;
7. Complete to 10 mL with Washing buffer A. Mix briefly;
8. Centrifuge for 3 minutes at 1500 g (4°C) in a swing-out rotor. Carefully discard the supernatant;
9. Re-suspend beads in 10mL of Washing buffer B. Briefly mix;
10. Centrifuge for 3 minutes at 1500 g (4°C) in a swing-out rotor. Carefully discard the supernatant;
11. Re-suspend the Sepharose beads in 10mL of Washing buffer C;
12. Centrifuge for 3 minutes at 1500 g (4°C) in a swing-out rotor. Carefully discard the supernatant;
13. To elute the chromatin, re-suspend the Sepharose beads in 500µl of Elution buffer and transfer to a 1.5mL Eppendorf tube;
14. Incubate for ~30 minutes at room temperature on a rotating wheel at 20-30 rpm. After this incubation, centrifuge for 3 minutes at 1500 rpm in a bench-top micro-centrifuge;
15. Carefully transfer the supernatant into a 2mL Eppendorf tube. The supernatant contains the chromatin eluted from the Sepharose beads (i.e. the bound fraction). To be stored on ice.

DNA extraction and Assessment of Precipitated Chromatin

DNA Extraction from Precipitated Chromatin

1. Add 500µL of phenol:chloroform:iso-amylalcohol 25:24:1 (v/v/v) to bound (step 15 of previous section) and unbound (step 4 previous section) fractions;
2. Vortex 30 seconds;
3. Centrifuge at 13,000 rpm (~15,000 g) for 15 minutes in a bench-top micro-centrifuge;
4. Carefully transfer the upper, aqueous, phase to another 2mL Eppendorf tube;
5. Add NaCl to a final concentration of 250 mM;
6. Add 20-40µg of glycogen and mix. Since the DNA concentration in the bound fraction is usually low, we recommend the use of glycogen as co-precipitator. This step is not necessary for the 'unbound' fraction;
7. Add 1 volume of iso-propanol;
8. Mix and store at -80°C for at least 2 hours;
9. Centrifuge at 13,000 rpm in a micro-centrifuge for 15 minutes. Carefully discard the supernatants;
10. Rinse the pellets with 1mL of 70 % (v/v) ethanol;
11. Centrifuge at 13,000 rpm for 5 minutes in a micro-centrifuge. Carefully discard the supernatants;
12. Dry pellets for 5-10 minutes at room temperature and re-suspend them in 10-50µl of 1X TE buffer.